The immune system of a mammal is one of the most versatile biological systems as probably greater than 1×107 antibody specificities can be produced. In an individual animal there are at least 5,000-10,000 different B-cell clones capable of generating unique antibodies. Further, because of the process of somatic mutation during the generation of antibody diversity, essentially an unlimited number of unique antibody molecules may be generated. Indeed, much of contemporary biological and medical research is directed toward tapping this repertoire. The development of the hybridoma methodology by Kohler and Milstein made it possible to produce monoclonal antibodies, i.e., a composition of antibody molecules of a single specificity, from the repertoire of antibodies induced during an immune response.
Unfortunately, current methods for generating monoclonal antibodies are not capable of efficiently surveying the entire antibody response induced by a particular immunogen. In contrast to this vast potential for different antibodies, current hybridoma methodologies typically yield only a few hundred different monoclonal antibodies per fusion.
Other difficulties in producing monoclonal antibodies with the hybridoma methodology include genetic instability and low production capacity of hybridoma cultures. One means by which the art has attempted to overcome these latter two problems has been to clone the immunoglobulin-producing genes from a particular hybridoma of interest into an expression system.
Analysis of antibodies expressed at a given moment in a subject is not trivial, since the immunoglobulin repertoire probably contains millions of different molecules. Only a small number of reagents capable of specifically recognizing elements of such a repertoire are currently available. Determining the sequence of expressed genes is possible; however, practically speaking, it is difficult to analyze routinely more than about ten or, perhaps, a hundred genes, and the operation is expensive and time-consuming. In short, the repertoire of immunoglobulins is described at the present time only by a small number of means.
The present invention provides methods to efficiently produce high-affinity monoclonal antibodies taking advantage of natural diversity and high diversity approaches.